Dna ligation on rna template

ABSTRACT

Disclosed are methods and compositions for detection and amplification of nucleic acids, wherein two DNA strands hybridized to an RNA strand are ligated. In one aspect, the disclosed methods include removal of an energy source, such as ATP, upon charging a ligase to form an enzyme-AMP intermediate, and then adding substrate, which results in one complete round of RNA-templated DNA ligation. In another aspect, the ligation reaction is accomplished by use of a mixture of at least two different ligase enzymes. The disclosed methods and compositions for RNA-templated DNA ligation may be particularly useful for detection of RNA sequence variants, for example RNA splice variants, and for quantitative expression analysis.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/798,238filed Oct. 30, 2017, which is a division of U.S. application Ser. No.14/313,211 fled Jun. 24, 2014 now abandoned, which is a continuation ofU.S. application Ser. No. 12/690,101 filed on Jan. 19, 2010 now U.S.Pat. No. 8,790,873 issued on Jul. 29, 2014 which claims the priority ofU.S. Provisional Application No. 61/145,466 filed Jan. 16, 2009 thedisclosure of each of which is incorporated herein by reference in theirentirety for all purposes.

FIELD OF THE INVENTION

Disclosed are methods related generally to ligation-mediated nucleicacid detection and analysis. Such methods may be performed on a solidsupport. Disclosed are also methods of gene expression profiling andmethods of sequencing of RNA, mediated by ligase enzymes. The methods donot require an intermediate step of generating a cDNA copy of thesequenced RNA by RT-PCR.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted electronically is hereby incorporated byreference. The file is named 3865_1_ST25.txt, the file is 1.68 KB andthe date of creation is Jan. 19, 2010.

BACKGROUND OF THE INVENTION

Tools for high-throughput nucleic acid analysis are becomingincreasingly important in light of recent advancements in availabilityof nucleic acid sequence information and genomic data for humans andother organisms. The techniques of ligation-mediated detection ofnucleic acids, coupled with hybridization of nucleic acids on arrays arewidely used form the basis of genomics applications such asoligonucleotide ligation assay (OLA) (Landegren et al., Science,241:1077 (1998)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci.USA, 88:189 (1991)), and ligation of padlock or open circle probes(Nilsson et al., Science, 265:2085 (1994)). Ligation-mediated nucleicacid detection methodology relies on the ability of ligases toaccurately discriminate between highly homologous nucleotide sequences,differing in some instances only at the terminal nucleotide position.Thus far, most ligation applications involve DNA-templated DNA ligationby a DNA ligase, where both the target nucleic acid and theoligonucleotide probes consist of deoxyribonucleotide polymers. Suchapplications are particularly useful for generating genomic sequencedata and SNP profiling.

RNA-templated DNA ligation is an attractive method for detection of RNA,determination of RNA sequence identity, expression monitoring andtranscript analysis. Direct detection of RNA target-DNA probe duplexes(without first converting RNA to cDNA by reverse transcription) has beenchallenging because a majority of tested DNA ligases fail to ligatenicked DNA on an RNA template. The exception to this is T4 DNA ligase,which is able to ligate nicked DNA hybridized to a RNA strand at adepressed rate.

T4 DNA ligase is an enzyme belonging to the DNA ligase family of enzymes(E.C. 6.5.1.1) which catalyzes the formation of a covalentphosphodiester bond from a free 3′ hydroxyl group on one DNA moleculeand a free 5′ phosphate group of a second, separate DNA molecule, thuscovalently linking the two DNA strands together to form a single DNAstrand. This activity may also be applied to RNA and is especiallyuseful in molecular genetics where sticky (or blunt) ends ofdouble-stranded DNA (dsDNA) may be fused together with other dsDNAmolecules, both products of a restriction enzyme cut, for instance. DNAligases play critical roles in cell division, in a process calledlagging strand DNA replication, as well as cell recovery in the dsDNAbreak repair mechanism. DNA ligases also play critical roles in normalcellular processes used to generate diversity in the immune systempathways, i.e. during V(D)J recombination. Commercially exploited DNAligases include the bacteriophage T4 DNA ligase. T4 DNA ligase possessesthe basic activity of catalyzing formation of covalent phosphodiesterbonds, as described above, but only operates on double-strandedmolecules, i.e. DNA/DNA, DNA/RNA hybrids, and RNA/RNA. Like manyligases, T4 DNA ligase activity requires adenosine triphosphate (ATP) asa cofactor. Recombinant T4 DNA ligase, and various mutants thereof, iscommercially available.

The ligation reaction catalyzed by DNA ligase occurs in three generalsteps. First, the ligase enzyme is activated by charging with ATP.Addition of ATP to ligase enzyme causes formation of an intermediateAMP-enzyme species concomitant with hydrolysis of ATP to yield AMP.Second, the charged AMP-enzyme intermediate binds to the dsDNA (ordsRNA, or RNA/DNA complex) and transfers the AMP moiety to the free 5′terminal phosphate, to form a high energy 5′-5′ phosphate bond. Third,the enzyme provides the appropriate environment in which the 3′ hydroxylgroup of the second strand of DNA (or RNA) is able to attach the highenergy 5′-5′ phosphate bond, thereby forming a covalent phosphodiesterbond as a product and releasing ligase enzyme and AMP. Free enzyme doesnot bind the intermediate high energy 5′-5′ phosphate bond species to anappreciable amount. Thus, if the ligase prematurely releases from theduplex after formation of the high energy 5′-5′ phosphate bond, thereaction will typically end and the intermediate will not proceed to thefinal ligated product.

Methods are disclosed herein that provide for efficient RNA-templatedDNA ligation.

SUMMARY OF THE INVENTION

Methods for detection of nucleic acids involving ligation of DNA strandshybridized to an RNA strand are disclosed. Such methods are particularlyuseful, for example, in detection of RNA sequence variants and forquantitative expression analysis. Other plausible non-limiting usesinclude analysis of viral and ribosomal RNA.

The present methods involve charging T4 DNA ligase with ATP to form anAMP-enzyme intermediate complex. Excess ATP is then removed. Removal offree ATP after the charging step prevents premature cycling of T4 DNAligase and the accumulation of 5′ adenylated (unligatable) product. Thatis, during RNA-templated DNA ligation, under conditions where ATP ispresent, T4 DNA ligase will charge, as above, forming the AMP-enzymecomplex, then bind the RNA-DNA complex. This is typically followed bypremature release of the ligase from the nicked DNA, yielding DNA havinga high energy 5′-5′ phosphate bond on its free 5′ end, which is not asubstrate for the DNA ligase reaction. The remaining AMP on the 5′ endof the DNA nick prevents any further ligation attempts, since the highenergy 5′-5′ phosphate bond is not an efficient substrate for the ligaseenzyme. The cofactor ATP may be removed by either apyrase or hexokinasetreatment. Charging of the ligase in this manner, and addition of nearstoichiometric quantities of charged enzyme, allows the DNA ligaseenzyme to prepare the RNA-DNA complex for later RNA-templated ligationin an ATP-depleted solution.

Other methods disclosed herein rely on the use of a mixture of two DNAligase enzymes in the presence of ATP. The first ligase may be, forinstance, T4 DNA ligase, or any ligase which produces high energy 5′-5′phosphate bonds at the end of a nicked DNA molecule. The second ligasemay be selected from ligase enzymes, or mutants thereof, which arecapable of performing only the final step of ligation. In one aspect,the second ligase is a non-native enzyme.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the differences in DNA vs. RNA-templated ligationusing DNA ligase. DNA ligase is capable of efficiently ligating DNA-DNAhybridized complexes. In contrast. DNA ligase often prematurely releasesfrom an RNA-DNA complex after formation of a high energy 5′-5′ phosphatebond on the substrate, thus terminating the reaction for thatintermediate.

FIGS. 2A and 2B provide a schematic diagram outlining two methods forefficient RNA-templated DNA ligation, as disclosed in the presentinvention. FIG. 2A illustrates a method wherein ligase is first chargedwith ATP, to form a ligase-AMP species, ATP is then removed and theRNA-templated DNA introduced into the reaction, allowing for completionof one round of RNA-templated ligation. FIG. 2B illustrates a similarmethod in which RNA-templated DNA ligation is achieved using a mixtureof two different ligase enzymes.

FIG. 3 illustrates a step-by-step outline of a method whereinRNA-templated ligation of padlock probes is followed by PCR.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to exemplary embodiments. While thedisclosed methods and compositions will be described in conjunction withthe exemplary embodiments, it will be understood that these exemplaryembodiments are not intended to limit the invention. On the contrary,the disclosed methods and compositions are intended to encompassalternatives, modifications and equivalents, which may be includedwithin the spirit and scope of the present application.

The present methods and compositions relate to diverse fields impactedby the nature of molecular interaction, including chemistry, biology,medicine and diagnostics. Methods disclosed herein are advantageous infields, such as those in which genetic information is required quickly,as in clinical diagnostic laboratories or in large-scale undertakingssuch as the Human Genome Project.

The present methods and compositions have many embodiments and rely onmany patents, applications and other references for details known tothose of the art. Therefore, when a patent, application, or otherreference is cited or repeated below, it should be understood that theentire disclosure of the document cited is incorporated by reference inits entirety for all purposes as well as for the proposition that isrecited. All documents, i.e., publications and patent applications,cited in this disclosure, including the foregoing, are incorporatedherein by reference in their entireties for all purposes to the sameextent as if each of the individual documents were specifically andindividually indicated to be so incorporated herein by reference in itsentirety.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of the disclosed methods andcompositions may be presented in a range format. It should be understoodthat when a description is provided in range format, this is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the claimed invention(s). Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible sub-ranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6, etc., as well as individual numbers withinthat range, for example, 1, 2, 3, 4, 5, and 6. This understandingapplies regardless of the breadth of the range.

The present methods and compositions may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of one of skill in the art. Such conventionaltechniques include polymer array synthesis, hybridization, ligation, anddetection of hybridization using a detectable label. Specificillustrations of suitable techniques are provided by reference to theexample hereinbelow. However, other equivalent conventional proceduresmay also be employed. Such conventional techniques and descriptions maybe found in standard laboratory manuals, such as Genome Analysis: ALaboratory Manual Series (Vols. I-IV), Using Antibodies: A LaboratoryManual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, andMolecular Cloning: A Laboratory Manual (all from Cold Spring HarborLaboratory Press), Stryer, L. (1995), Biochemistry, 4th Ed., Freeman,New York, Gait, Oligonucleotide Synthesis: A Practical Approach, (1984),IRL Press, London, Nelson and Cox (2000), Lehninger, Principles ofBiochemistry, 3^(rd) Ed., W.H. Freeman Pub., New York, N.Y., and Berg etal. (2002), Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y.and Berg et al. (2002) Biochemistry, 5^(th) Ed., W.H. Freeman Pub., NewYork, N.Y., all of which are herein incorporated in their entirety byreference for all purposes.

The presently disclosed methods and compositions may employ solidsubstrates, including arrays in some embodiments. Methods and techniquesapplicable to polymer (including protein) array synthesis have beendescribed in U.S. Ser. No. 09/536,841 (abandoned), WO 00/58516, U.S.Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261,5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681,5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659,5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601,6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, andin PCT Applications Nos. PCT/US99/00730 (International Publication No.WO 99/36760) and PCT/US01/04285 (International Publication No. WO01/58593), which are all incorporated herein by reference in theirentirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present methods andcompositions include, but are not limited to, those that arecommercially available from Affymetrix (Santa Clara, Calif.) under thebrand name GENECHIP

. Example arrays are shown on the website at affymetrix.com.

Many uses for polymers attached to solid substrates are contemplatedherein. These uses include, but are not limited to, gene expressionmonitoring, profiling, library screening, genotyping and diagnostics.Methods of gene expression monitoring and profiling are described inU.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138,6,177,248 and 6,309,822. Genotyping and uses therefore are shown in U.S.patent application Ser. No. 10/442,021 (abandoned) and U.S. Pat. Nos.5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947, 6,368,799,6,333,179, and 6,872,529. Other uses are described in U.S. Pat. Nos.5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

Contemplated herein are embodiments employing various sample preparationmethods. Prior to, or concurrent with, genotyping, the genomic samplemay be amplified by a variety of mechanisms, some of which may employPCR. (See, for example, PCR Technology: Principles and Applications forDNA Amplification, Ed. H. A. Erlich, Freeman Press, NY, NY, 1992; PCRProtocols: A Guide to Methods and Applications, Eds. Innis, et al.,Academic Press, San Diego, Calif., 1990; Mattila et al., Nucleic AcidsRes., 19:4967, 1991; Eckert et al., PCR Methods and Applications, 1:17,1991; PCR, Eds. McPherson et al., IRL Press, Oxford, 1991; and U.S. Pat.Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675, each ofwhich is incorporated herein by reference in their entireties for allpurposes. The sample may also be amplified on the array. (See, forexample, U.S. Pat. No. 6,300,070 and U.S. patent application Ser. No.09/513,300 (abandoned), all of which are incorporated herein byreference).

Other suitable amplification methods include the ligase chain reaction(LCR) (see, for example, Wu and Wallace, Genomics, 4:560 (1989),Landegren et al., Science, 241:1077 (1988) and Barringer et al., Gene,89:117 (1990)), transcription amplification (Kwoh et al., Proc. Natd.Acad. Sci. USA, 86:1173 (1989) and WO 88/10315), self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA. 87:1874 (1990)and WO 90/06995), selective amplification of target polynucleotidesequences (U.S. Pat. No. 6,410,276), consensus sequence primedpolymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975),arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos.5,413,909 and 5,861,245) and nucleic acid based sequence amplification(NABSA). (See also, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603,each of which is incorporated herein by reference). Other amplificationmethods that may be used are described in, for instance, U.S. Pat. Nos.6,582,938, 5,242,794, 5,494,810, and 4,988,617, each of which isincorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch, 11:1418 (2001), U.S. Pat. Nos. 6,361,947, 6,391,592,6,632,611, 6,872,529 and 6,958,225, and in U.S. patent application Ser.No. 09/916,135 (abandoned).

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith known general binding methods, including those referred to inManiatis et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Ed.,Cold Spring Harbor, N.Y, (1989); Berger and Kimmel, Methods inEnzmology, Guide to Molecular Cloning Techniques, Vol. 152, AcademicPress, Inc., San Diego, Calif. (1987); Young and Davism, Proc. Nat'l.Acad. Sci., 80:1194 (1983). Methods and apparatus for performingrepeated and controlled hybridization reactions have been described in,for example, U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996, 6,386,749,and 6,391,623 each of which are incorporated herein by reference.

Presently contemplated are also methods and compositions employingsignal detection of hybridization between ligands. See U.S. Pat. Nos.5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956;6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,225,625, inU.S. Ser. No. 10/389,194 and in PCT Application PCT/US99/06097(published as WO99/47964), each of which also is hereby incorporated byreference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 10/389,194 (U.S.Patent Application Publication 20040012676), 60/493,495 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety for all purposes.

Embodiments may also employ conventional biology methods, software andsystems. Computer software products contemplated herein typicallyinclude computer readable medium having computer-executable instructionsfor performing the logic steps of the presently disclosed methods.Suitable computer readable medium include floppy disk,CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetictapes etc. The computer executable instructions may be written in asuitable computer language or combination of several languages. Basiccomputational biology methods are described in, for example Setubal andMeidanis et al., Introduction to Computational Biology Methods (PWSPublishing Company, Boston, 1997); Salzberg, Searles, Kasif, (Ed.),Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998);Rashidi and Buehler, Bioinformatics Basics: Application in BiologicalScience and Medicine (CRC Press, London, 2000) and Ouelette and BzevanisBioinformatics: A Practical Guide for Analysis of Gene and Proteins(Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S. Pat. No. 6,420,108.

The presently disclosed methods may also make use of various computerprogram products and software for a variety of purposes, such as probedesign, management of data, analysis, and instrument operation. (See,U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454,6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170).

Additionally, encompassed herein are embodiments that may includemethods for providing genetic information over networks such as theinternet, as disclosed in, for instance, U.S. patent application Ser.No. 10/197,621 (U.S. Patent Application Publication No. 20030097222),Ser. No. 10/063,559 (U.S. Patent Application Publication No.20020183936, abandoned), Ser. No. 10/065,856 (U.S. Patent ApplicationPublication No. 20030100995, abandoned), Ser. No. 10/065,868 (U.S.Patent Application Publication No. 20030120432, abandoned), Ser. No.10/328,818 (U.S. Patent Application Publication No. 20040002818,abandoned), Ser. No. 10/328,872 (U.S. Patent Application Publication No.20040126840, abandoned), Ser. No. 10/423,403 (U.S. Patent ApplicationPublication No. 20040049354, abandoned), and 60/482,389 (expired).

A. Definitions

The term “apyrase,” as used herein, refers to one or more of acalcium-activated enzyme which possesses ATP-diphosphohydrolase activityand catalyzes the hydrolysis of the gamma phosphate from ATP, andcatalyzes the hydrolysis of the beta phosphate from ADP. Apyrases arefound in all eukaryotes and some prokaryotic organisms, indicating apreserved role for these enzymes across species. They a distinctphosphohydrolase activity, nucleotide substrate specificity, divalentcation requirement, and sensitivity to inhibitors. (See, Plesner, Int.Rev. Cytol., 158:141 (1995), and Handa and Guidotti, Biochem. Biophys.Res. Commun., 218(3):916 (1996)). In mammals, apyrase is believed tofunction primarily as an extracellular hydrolase specific for ATP andADP, which function is important in the inactivation of synaptic ATPmolecules following nerve stimulation. (See, Todorov et al., Nature,387(6628):76 (1997)). Apyrase in mammals is also believed to beimportant in the inhibition of ADP-induced platelet aggregation. (See,Marcus et al., J. Clin. Invest., 99(6):1351 (1997)). Recombinant apyraseis commercially available from New England Biolabs.

The term “array” as used herein refers to an intentionally createdcollection of molecules which can be prepared either synthetically orbiosynthetically. The molecules in the array can be identical ordifferent from each other. The array can assume a variety of formatsincluding, but not limited to, libraries of soluble molecules, andlibraries of compounds tethered to resin beads, silica chips, or othersolid supports.

The term “complementary” refers to the hybridization or base-pairingbetween nucleotides or nucleic acids, such as, for instance, that whichoccurs between the two strands of a double stranded DNA molecule, orbetween an oligonucleotide primer and a primer binding site on a singlestranded nucleic acid to be sequenced or amplified. Complementarynucleotides are pairs of nucleotides having the following identities,generally, A and T (or A and U), or C and G. Two single stranded RNA orDNA molecules are said to be complementary when the nucleotides of onestrand, optimally aligned and compared, taking into considerationvarious nucleotide insertions or deletions, if present, pair with atleast about 80% of the nucleotides of the other strand, usually at leastabout 90% to 95%, and more preferably from about 98 to 100%.Alternatively, complementarity exists when an RNA or DNA polynucleotidewill hybridize under selective (or stringent) hybridization conditionsto its complement. Typically, selective hybridization will occur whenthere is at least about 65% complementary over a stretch of at least 14to 25 nucleotides, preferably at least about 75%, more preferably atleast about 90%, or 95%, complementary.

The term “genome” as used herein includes all of the genetic material inthe chromosomes of an organism. DNA obtained from the genetic materialin the chromosomes of a particular organism is genomic DNA. A genomiclibrary is a collection of clones made from a set of randomly generatedoverlapping DNA fragments representing the entire genome of an organism.

The term “genotyping” refers to the determination of the geneticinformation an individual carries at one or more positions in thegenome. For example, genotyping may comprise the determination of whichallele or alleles an individual carries for a single SNP or thedetermination of which allele or alleles an individual carries for aplurality of SNPs. For example, a particular nucleotide in a genome maybe an A in some individuals and a C in other individuals. Thoseindividuals who have an A at the position have the A allele and thosewho have a C have the C allele. In a diploid organism the individualwill have two copies of the sequence containing the polymorphicposition. Thus, the individual may have an A allele and a C allele oralternatively two copies of the A allele or two copies of the C allele.Those individuals who have two copies of the C allele are homozygous forthe C allele. Individuals who have two copies of the A allele arehomozygous for the A allele. Individuals who have one copy of eachallele are heterozygous. The array may be designed to distinguishbetween each of these three possible outcomes. A polymorphic locationmay have two or more possible alleles and the array may be designed todistinguish between all possible combinations.

The term “hexokinase” refers to a family of enzymes that catalyze thephosphorylation of a six-carbon sugar, a hexose, to yield a hexosephosphate as the product, in the presence of ATP. Hexokinase enzymes areubiquitously expressed in eukaryotes and prokaryotes, with severalisoforms of the enzyme often found within a single species. Thedifferent hexokinase family members share a common ATP-binding site coresurrounded by variable domains that determine substrate specificity andother functions, e.g. subcellular localization. There are four mammalianhexokinases, numbered I-IV. Mammalian hexokinase IV (glucokinase) playsa key role in the regulation of glucose metabolism and homeostasis.Hexokinase from S. cerevisiae is commercially available, for example,from USB and Sigma-Aldrich.

The term “hybridization” as used herein refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide; triple-stranded hybridization is alsotheoretically possible. The resulting (usually) double-strandedpolynucleotide is a “hybrid.” Hybridizations are usually performed understringent conditions, for example, at a salt concentration of no morethan 1 M and a temperature of at least 25° C. For example, conditions of5×SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and atemperature of between 25° C. and 30° C. are suitable forallele-specific probe hybridizations. Hybridization conditions generallysuitable for microarrays are described in the Gene Expression TechnicalManual, 2004, and the GeneChip Mapping Assay Manual, 2004, available atAffymetrix.com.

The term “label” as used herein refers to, but is not limited to, aluminescent label, a light scattering label or a radioactive label.Fluorescent labels include, inter alia, the commercially availablefluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite(Millipore) and FAM (ABI). (See, U.S. Pat. No. 6,287,778, incorporatedherein by reference).

The term “DNA ligase,” as used herein, refers to a family of enzymeswhich catalyze the formation of a covalent phosphodiester bond betweentwo distinct DNA strands, i.e. a ligation reaction. Two prokaryotic DNAligases, namely the ATP-dependent T4 DNA ligase (isolated from the T4phage), and the NAD⁺-dependent DNA ligase from E. coli, have becomeindispensable tools in molecular biology applications. Both enzymescatalyze the synthesis of a phosphodiester bond between the 3′-hydroxylgroup of one polynucleic acid, and the 5′-phosphoryl group, of a secondpolynucleic acid, for instance at a nick between the two strands whichare both hybridized to a third DNA strand. The mechanism of the ligationreaction catalyzed by this family of enzymes typically requires threeenzymatic steps. The initial step involves attack of the α-phosphorylgroup of either ATP or NAD⁺, resulting in formation of aligase-adenylate intermediate (AMP is covalently linked to a lysineresidue of the enzyme), and concurrent release of either pyrophosphate(PP_(i)) or nicotinamide mononucleotide (NAD⁺). In the second step ofthe enzymatic reaction, AMP is transferred to the 5′ end of the free 5′phosphate terminus of one DNA strand, to form an intermediate species ofDNA-adenylate. In the final step, ligase catalyzes the attack of theDNA-adenylate intermediate species by the 3′ hydroxyl group of thesecond DNA strand, resulting in formation of a phosphodiester bond andsealing of the nick between the two DNA strands, and concurrent releaseof AMP. RNA ligases, which are a related family of enzymes, catalyze theligation of nicked RNA ends hybridized on to RNA or DNA in an analogousfashion. T4 DNA ligase is commercially available from at least USB andNew England Biolabs.

The terms “mRNA” and “mRNA transcripts,” as used herein, include, butare not limited to, pre-mRNA transcript(s), transcript processingintermediates, mature mRNA(s) ready for translation and transcripts ofthe gene or genes, or nucleic acids derived from the mRNA transcript(s).Transcript processing may include splicing, editing and degradation. Asused herein, a nucleic acid derived from an mRNA transcript refers to anucleic acid for whose synthesis the mRNA transcript or a subsequencethereof has ultimately served as a template. Thus, a cDNA reversetranscribed from an mRNA, an RNA transcribed from that cDNA, a DNAamplified from the cDNA, an RNA transcribed from the amplified DNA,etc., are all derived from the mRNA transcript, and detection of suchderived products is indicative of the presence and/or abundance of theoriginal transcript in a sample. Thus, mRNA derived samples include, butare not limited to, mRNA transcripts of the gene or genes, cDNA reversetranscribed from the mRNA, cRNA transcribed from the cDNA, DNA amplifiedfrom the genes, RNA transcribed from amplified DNA, and the like.

The term “nucleic acid” as used herein refers to a polymeric form ofnucleotides of any length, either ribonucleotides, deoxyribonucleotidesor peptide nucleic acids (PNAs), that comprise purine and pyrimidinebases, or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases. The backbone of thepolynucleotide can comprise sugars and phosphate groups, as maytypically be found in RNA or DNA, or modified or substituted sugar orphosphate groups. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs. The sequence of anucleic acid may be interrupted by non-nucleotide components. Thus theterms nucleoside, nucleotide, deoxynucleoside and deoxynucleotidegenerally include analogs such as those described herein. These analogsmay possess structural features that are common with a naturallyoccurring nucleoside or nucleotide, such that when incorporated into anucleic acid or oligonucleoside sequence, the analogs allowhybridization of the nucleic acid with a naturally occurring nucleicacid sequence in solution. Typically, such analogs are derived fromnaturally occurring nucleosides and nucleotides by replacing and/ormodifying the base, the ribose, or the phosphodiester moiety. Thesechanges incorporated into the analogs can be specifically designed, andtherefore function, to stabilize or destabilize hybrid formation, orenhance the specificity of hybridization with a complementary nucleicacid sequence, as desired.

The terms “oligonucleotide” and “polynucleotide” as used herein refer toa nucleic acid ranging from at least 2, preferably at least 8, and morepreferably at least 20 nucleotides in length or a compound thatspecifically hybridizes to a polynucleotide. Polynucleotidescontemplated herein include sequences of deoxyribonucleic acid (DNA) orribonucleic acid (RNA) which may be isolated from natural sources,recombinantly produced or artificially synthesized and mimetics thereof.Further examples of such polynucleotides may be peptide nucleic acid(PNA). Also contemplated are embodiments in which there is anontraditional base pairing such as Hoogsteen base pairing which hasbeen identified in certain tRNA molecules and postulated to exist in atriple helix. “Polynucleotide” and “oligonucleotide” are usedinterchangeably in this application.

The term “polymorphism” as used herein refers to the occurrence of twoor more genetically determined alternative sequences or alleles in apopulation. A polymorphic marker or site is the locus at whichdivergence occurs. Preferred markers have at least two alleles, eachoccurring at frequency of greater than 1%, and more preferably greaterthan 10% or 20% of a selected population. A polymorphism may compriseone or more base changes, an insertion, a repeat, or a deletion. Apolymorphic locus may be as small as one base pair. Polymorphic markersinclude restriction fragment length polymorphisms, variable number oftandem repeats (VNTR's), hypervariable regions, minisatellites,dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats,simple sequence repeats, and insertion elements such as Alu. The firstidentified allelic form is arbitrarily designated as the reference formand other allelic forms are designated as alternative or variantalleles. The allelic form occurring most frequently in a selectedpopulation is sometimes referred to as the wild type form. Diploidorganisms may be homozygous or heterozygous for allelic forms. Abiallelic polymorphism has two forms. A triallelic polymorphism hasthree forms. Single nucleotide polymorphisms (SNPs) are included inpolymorphisms. Single nucleotide polymorphisms (SNPs) are positions atwhich two alternative bases occur at appreciable frequency (>1%) in agiven population. SNPs are the most common type of human geneticvariation. A polymorphic site is frequently preceded by and followed byhighly conserved sequences (e.g., sequences that vary in less than 1/100or 1/1000 members of the populations).

A SNP may arise due to substitution of one nucleotide for another at thepolymorphic site. A transition is the replacement of one purine byanother purine or one pyrimidine by another pyrimidine. A transversionis the replacement of a purine by a pyrimidine or vice versa. SNPs canalso arise from a deletion of a nucleotide or an insertion of anucleotide relative to a reference allele.

The term “probe” as used herein refers to a surface-immobilized orfree-in-solution molecule that can be recognized by a particular target.U.S. Pat. No. 6,582,908 provides an example of arrays having allpossible combinations of nucleic acid-based probes having a length of 10bases, and 12 bases or more. In one embodiment, a probe may consist ofan open circle molecule, comprising a nucleic acid having left and rightarms whose sequences are complementary to the target, and separated by alinker region. Open circle probes are described in, for instance, U.S.Pat. No. 6,858,412, and Hardenbol et al., Nat. Biotechno., 21(6):673(2003). In another embodiment, a probe, such as a nucleic acid, may beattached to a microparticle carrying a distinguishable code. Encodedmicroparticles which can be used for in-solution array assays aredescribed in, for instance, International Patent Application PublicationNo. WO 2007/081410, and U.S. patent application Ser. No. 11/521,057(U.S. Patent Application Publication No. 2008/0038559). Examples ofnucleic acid probe sequences that may be investigated using thedisclosed methods and compositions include, but are not restricted to,those that are complementary to genes encoding agonists and antagonistsfor cell membrane receptors, toxins and venoms, viral epitopes, hormones(for example, opioid peptides, steroids, etc.), hormone receptors,peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, proteins, andmonoclonal antibodies.

The term “solid support”, “support”, and “substrate” as used herein areused interchangeably and refer to a material or group of materialshaving a rigid or semi-rigid surface or surfaces. In many embodiments,at least one surface of the solid support will be substantially flat,although in some embodiments it may be desirable to physically separatesynthesis regions for different compounds with, for example, wells,raised regions, pins, etched trenches, or the like. According to otherembodiments, the solid support(s) will take the form of beads, resins,gels, microspheres, or other geometric configurations. (See, U.S. Pat.No. 5,744,305 for exemplary substrates).

The term “target” as used herein refers to a molecule that has anaffinity for a given probe. Targets may be naturally occurring orman-made, i.e. synthetic, molecules. Targets may be employed in theirunaltered state, or as aggregates with other species. Targets may beattached, covalently or noncovalently, to a binding member, eitherdirectly or via a specific binding substance. Examples of targets whichcan be employed herein include, but are not restricted to, antibodies,cell membrane receptors, monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells or othermaterials), drugs, oligonucleotides, nucleic acids, peptides, cofactors,lectins, sugars, polysaccharides, cells, cellular membranes, and cellorganelles. Targets are sometimes referred to in the art as anti-probes.As the term “targets” is used herein, no difference in meaning betweenthis term and the term “anti-probe” is intended. A “probe-targetcomplex” is formed when two macromolecules have combined throughmolecular recognition to form a complex.

B. RNA-Templated Ligation

Many disease states are characterized by differences in the expressionlevels of various genes, either through transcriptional regulation orthrough copy number changes at the level of genomic DNA. A common methodof monitoring gene expression is to detect and quantitate specific RNAtranscripts. This may be accomplished by employing microarraytechnology. However, closely related RNA sequences pose a challenge forexpression analysis using microarray technology because the expressionof, and quantity of, mRNA of different genes vary dramatically and undercertain conditions, can compete with each other for probe hybridization.In other words, related RNA sequences that are not perfectlycomplementary to the probe sequence, but which are expressed at a muchhigher level than the target RNA, can potentially hybridize to the probeunder standard hybridization conditions, thereby skewing results ofhybridization-based assays. In addition to mRNA transcript detection andanalysis, the disclosed methods can be used for detection of allribonucleic acids from all sources (for example, ribosomal RNA and viralRNA).

Further, it is clear that genotyping of polymorphisms in RNA, such assingle nucleotide polymorphisms (SNPs), provides useful information inanalysis of disease physiology and progression. That is, alternativesplicing at the mRNA level is known to lead to different translationproducts that possess different activities. These differences maycorrelate to various disease symptoms. Thus, research is now beingdirected at detecting various alternative splicing events. The abilityto detect and quantitate such alternative splicing events may beachieved through the following methods and compositions. For instance,in one embodiment, DNA probes may be immobile onto an array. The DNAprobes could be complementary to, for instance the last(downstream-most) exon of an mRNA molecule. All mRNA transcriptspossessing that exon will hybridize to the immobilized DNA probe.Following this hybridization, a second DNA probe may be added which iscomplementary to one of several possible alternative splice sequences inthe mRNA, upstream of the sequence hybridized to the immobilized probe.The mRNA transcript will now be hybridized to, and serving as a templatefor, the two DNA probes, one of which is immobilized. DNA polymerase maythen be used to ligate the two probes together, if the second DNA probehybridizes to the mRNA transcript. The second DNA probe could include adetectable label. Ligation of the second probe to the first probe willtherefore leave a detectable label covalently bound to the array andafter washing steps, it will be detected, thus indicating the presenceof the alternative splice variant.

One of skill in the art will be familiar with such methodologies andunderstand that many variations exist on this type of assay. Forinstance, molecular inversion probes (MIPs) may also be used in thismanner to detect mRNA splice variants. Such assays may even be performeddirectly on Formalin-Fixed, Paraffin-Embedded (FFPE) samples orfresh-frozen samples.

DNA ligases are able to distinguish single nucleotide variations amongDNA sequences and therefore may be exploited in such expressionprofiling analyses. DNA ligases are relatively inefficient in ligatingtwo nucleic acid sequences possessing non-complementary termini. Thisproperty of the ligase enzyme has been exploited in an assay termed theoligonucleotide ligation assay (OLA). (See, Landegren et al., Science,241:1077 (1998); Wu, D. Y. & Wallace, R. B. Gene 76, 245-254 (1989);Luo, J., Bergstrom, D. E. & Barany, F., Nucleic Acids Res. 24, 3071-3078(1996); and Tong, J., Cao, W. & Barany, F., Nucleic Acids Res. 27,788-794 (1999)). Another method exploiting this property of the ligaseis termed the ligase chain reaction. (See, Barany, Proc. Nat'l. Acad.Sci. USA, 88:189 (1991)). However, direct detection of RNA target-DNAprobe duplexes (without first converting RNA to cDNA by reversetranscription) is difficult given that a majority of DNA ligases fail toligate nicked DNA hybridized to an RNA template.

Presently, only T4 DNA ligase has been shown to catalyze the ligation ofDNA oligonucleotides hybridized to RNA templates. However, this functionof T4 DNA ligase is substantially reduced in catalytic efficiency ascompared to its catalyzation of DNA-templated reactions. (See, Kleppe etal., Proc. Nat'l. Acad. Sci. US4, 67:68 (1970), and Fareed et al., J.Biol. Chem., 246:925 (1971)).

It has previously been reported that under low salt, particularlymonovalent cations, and low ATP concentrations, high concentrations ofT4 DNA ligase efficiently join DNA oligonucleotides hybridized injuxtaposition on RNA target strands. See, US Patent Pub. 20070225487,which is incorporated herein by reference in its entirety.

It is also known that some of the ATP dependent DNA ligases ineukaryotes and eucaryotic virus can use RNA as a template for DNAligation (see, Tomkinson, A. E. and Mackey, Z. B. Mutation Res. 407 1-9(1998), Sekiguchi, J. and Shuman, S. Biochemistry 36 9073-9079 (1997),and Sriskanda, V. and Shuman, S. Nucleic Acids Res. 26 3536-3541(1998)). However, it is not know how efficient the reaction is usingthese enzymes, and eukaryotic enzymes have not been used for geneanalytic assays. Also ATP-dependent DNA ligases from thermophilicarcheon such as Methanobacterium thermoautotrophicum (Sriskanda, V., etal. Nucleic Acids Res 28 2221-2228. (2000)) can join DNAoligonucleotides. The general features of the detection methodsdescribed herein should be generally applicable for all DNA ligases.

RNA-templated ligation of DNA probes has been used to generatemolecules, amplifiable by PCR via general sequences present at theremote ends of a pair of ligation probes (Hsuih, T. C. H. et al. J.Clin. Microbiol. 34, 501-507 (1996). The method has been applied todetect viral RNA extracted from clinical and archival specimens withincreased sensitivity compared to nested RT-PCR (Park, Y. N. et al., Am.J Pathology 149, 1485-1491 (1996); Miyauchi, I., Moriyama, M., Zhang, D.Y. & Abe, K., Path. Int. 48, 428-432 (1998)). RNA-templated ligation ofRNA probes has been used for detection of transcripts in experimentswhere ligation products were amplified by the Qb replicase (Tyagi, S.,Landegren, U., Tazi, M., Lizardi, P. M. & Kramer, F. R., Proc. Natl.Acad. Sci. USA 93, 5395-5400 (1996). RNA-templated ligation of eitherDNA or RNA probes can thus substitute for a reverse transcription (RT)step before amplification.

T4 DNA ligase catalyzes DNA ligation in three steps. First, the enzymeis activated through ATP hydrolysis, resulting in covalent addition ofAMP to the enzyme. This intermediate enzyme species then binds to anicked DNA site. Second, after binding to the nicked DNA site, theligase transfers AMP to the phosphate at the 5′ end of the nick, formingan intermediate product possessing a 5′ to 5′ pyrophosphate bond. Third,the ligase catalyzes formation of a phosphodiester bond by attack of theintermediate 5′ to 5′ pyrophosphate bond by the OH group of the 3′ endof the second strand of DNA, resulting in release of the ligase and AMP.

In RNA-templated ligation, T4 DNA ligase will prematurely release fromthe nicked DNA before the final step, leaving the 5′ to 5′ pyrophosphatebond intermediate species. Released T4 DNA ligase then re-charges itselfusing ATP, preventing the enzyme-AMP intermediate species fromcompleting the final step of the reaction, ultimately yielding anaccumulation of 5′ to 5′ pyrophosphate bond intermediate product. It hasbeen shown that incubation of the substrate with T4 DNA ligase at a verylow ATP concentration improves the ligation efficiency of T4 DNA ligase,but the reaction proceeds very slowly and requires relatively largeamounts of enzyme. (See, Nilsson et al., Nucleic Acids Res., 29(2):578(2001). Moreover, the longer (2 hour or more) incubation period combinedwith the necessary determination of an optimum ATP concentration can bean obstacle for a number of applications.

Although RNA-templated DNA ligation promises to be an attractive methodfor expression monitoring and transcript analysis, the poor ligationefficiency of T4 DNA ligase on RNA templates has hampered thedevelopment of methodologies relying on ligation-mediated detection andanalysis of ribonucleic acids. Methods are disclosed that provide forefficient RNA-templated DNA ligation.

In one embodiment, as depicted in FIG. 2a , the disclosed methodsinvolve charging T4 DNA ligase with ATP to form the charged enzyme-AMPspecies, followed by removal of excess or unused ATP to preventpremature cycling of T4 DNA ligase and the accumulation of the 5′ to 5′pyrophosphate bond intermediate product. Removal of free ATP by eitheraddition of apyrase or hexokinase, under proper buffer conditions, forinstance, pushes the reaction in the direction of a single round of T4DNA ligase-mediated ligation. Of course, any known method of ATPdepletion may be employed for this purpose. Depletion of ATP aftercharging of the enzyme, by any means which does not further interferewith the ligation reaction, will be suitable in the present method.

In another embodiment, depicted in FIG. 2b , the disclosed methods mayemploy a mixture of at least two ligases in the presence of ATP. In thisembodiment, the first ligase may be, for instance, T4 DNA ligase, oranother ligase capable of producing 5′ to 5′ pyrophosphate bond productintermediates. The second ligase employed in this embodiment may be anyof a number of mutant forms of ligase which catalyze the final step ofligation, i.e. formation of the desired phosphodiester bond between thetwo strands of DNA template on the RNA molecule, by consumption of the5′ to 5′ pyrophosphate bond on the terminus of the first strand of DNAand the terminal hydroxyl group from the second strand of DNA. Mutantligases that are unable to charge with AMP, i.e. form the intermediateenzyme-AMP complex, but retain the catalytic activity of formation ofthe covalent phosphodiester bond between the two DNA strands have beendescribed. Such mutant ligase enzymes include, but are not limited to,several point mutants and amino-terminal truncation mutants of the E.coli NAD⁺-dependent DNA ligase. (See, Sriskanda and Shuman, J. Biol.Chem., 277:9685 (2002)). In this embodiment, multiple different ligasesmay be utilized for the second ligase, thus the reaction may comprise asmany as three different ligases, or four different ligases, or fivedifferent ligases, or more. The number of ligases utilized in thereaction mixture is not constrained, so long as the ligases possess theactivity required to complete the reaction.

The following list of examples is provided for illustrative purposesonly. While the present disclosure is intended to encompass theseexamples, it will be clear to one of skill in the art that these arenon-limiting examples and that many modifications may be made to theexamples while still maintaining subject matter within the scope of thepresent application. For instance, the following methods may be utilizedin a series of steps, comprising intermediate steps, steps before andafter the provided methods, which further treat and/or prepare thenucleic acids utilized therein. For instance, nucleic acids used hereinmay also be concurrently or subsequently utilized in microarrayanalyses, further PCR or transformation reactions, etc. Therefore, theseexamples are non-limiting.

Detection of ligation may be conducted by many means. For instance,detectable labels may be added to substrate nucleotides. Theincorporation of these detectable labels into the ligated product may bemeasured. Detectable labels may include, but are not limited to, use offluorescent labels, radioactive labels, phosphorescent labels, etc.Ligation may also be measured by detecting a shift in the substrate bandin a gel-shift assay. There exist many means of detecting such ligationevents. One of skill in the art will know how to modify and incorporatesuch means of detection in the context of the present methods andreagents. Such means of detection are incorporated herein for thepurpose of measuring the ligation event.

EXAMPLES Example 1 RNA-Templated Ligation Using Hexokinase Treatment toEliminate Free ATP

Experimental design involves use of a padlock-type assay wherelinearized DNA is annealed at two positions onto a linearized RNAtemplate. Padlock probe assays, design and methodologies are disclosedin, for instance, U.S. Pat. Nos. 5,871,921, 6,235,472, 5,866,337, andJapanese patent JP 4-262799, the disclosures of which are incorporatedherein by reference in their entirety. Thus, when DNA polymerase isadded, and the assay is successful, the enzyme ligates the two ends ofthe DNA, forming a circularized, single-stranded DNA molecule which maybe assayed by gel shift assay or any other means.

In the present experiment, RNA template was prepared from linearizedTch2, H4 or H5 plasmid DNA by in vitro transcription using Ambion'sMegascript kit. The TCH2 gene is an Arabidopsis thaliana gene encoding acalcium ion binding protein. Four padlock probes (probe sequences may befound in Table 1) were designed, having the sequences shown in Table 1.Thus, in the 5′ to 3′ direction, the sequence for the first padlockwould be as follows: 5′-probe 1b-bridge-probe 1a-3′. Probes 2-4 aresimilarly arranged in sequence. As is customary in MIP probes, thebridging sequences may comprise the following elements: a firstuniversal PCR primer site, a depyrimidination site (to invert and/orlinearize the MIP probe) including the sequence UUU as a uracil DNAglycosylase site, a second universal PCR primer site, a detectablylabeled nucleotide for quantitation, a barcode/tag sequence, a DraIrestriction site (or any restriction site to release the barcodesequence).

TABLE 1 SEQ Probe ID # # NO. Sequences bp 1a 1GGCCAGTGCTGGAGTTCGCACGCTATATTTAA 53 AAGCATCACCAGAAGAAACAG 1b 2TAACGATGATGAAACAATTCGACCTGTCCACG 32 2a 3GGCCAGTGCTGGAGTTCGCACGCTATATTTAA 53 ATCAACAATGTCATCGAAGAA 2b 4AAATCTCCGTCGACGAGCTCGTCCACG 27 3a 5 GGCCAGTGCTGGAGTTCGCACGCTATATTTAA 53AGGACGACATCAAAAAAGTCT 3b 6 TCCAACGATTCGACAAAAACGTCCACG 27 4a 7GGCCAGTGCTGGAGTTCGCACGCTATATTTAA 53 AAAATCTCCGTCGACGAGCTC 4b 8AAAGAAGTGATCCGCGCTCTGTCCACG 27

T4 DNA ligase was charged for 20 minutes at room temperature in thepresence of ATP. ATP depletion mix was added to the charging mix and thereaction proceeded for another 30 minutes at 37° C. to remove remainingfree ATP. In the meantime, RNA/probe mixes were heated to 80° C. for 3minutes, cooled to 46° C., and then RNAse inhibitors were added followedby 20-minute incubation at 46° C. 5 μl of RNA/probe mix was combinedwith 5 μl of charged ligase mix, and incubated at 37° C. for 30 minutes,1 hour, or 2 hours. Ligation reactions were stopped by adding an equalamount of formamide stop solution, and heating to 95° C. for 3 minutes,followed by cooling on ice. The ligated samples were loaded onto an 8%sequencing gel, and resolved ligation products were visualized by SYBRgreen or ethidium bromide staining. Table 2 provides buffercompositions.

TABLE 2 ATP/Ligase Charging Mix ATP Depletion Mix Vol Vol Ingredient(μl) Ingredient (μl) ATP (2 mM) 2.5 Hexokinase (10 U/μl) 2 T4 DNA ligase(2000 U/μl) 4 200 mM glucose 25 H₂O 2.5 RNAse inhibitor 2 10 X rxnbuffer 1 H₂O 6 10 X rxn buffer 5 Charging mix 10 Total 10 Total 50RNA/Probe 1 Mix RNA/Probe 2 Mix Vol Vol Ingredient (μl) Ingredient (μl)probe 1 (500 nM) 0.84 probe 2 (792 nM) 0.55 RNA (5.3 uM) 0.63 RNA (5.3uM) 0.63 RNAse inhibitor 3 RNAse inhibitor 3 H₂O 15.43 H₂O 15.74 Total21 Total 21

Example 2 RNA-Templated Ligation Using Apyrase Treatment to Deplete FreeATP

The experiment was conducted as described in Example 1, except that theATP depleting mix consisted of 1 μl of apyrase enzyme (500 mU/μl), 5 μlof 10× reaction buffer, and 34 μl of distilled water instead ofhexokinase enzyme. The ATP depleting mix was added to 10 μl of chargingmix, as in Example 1, and incubated for 30 minutes at room temperature.The ATP concentration in the charging mix was either 500 μM or 200 μM.

Example 3 RNA-Templated Ligation Using a Mixture of at Least Two Ligases

Charging of T4 ligase and ligation steps may be conducted as describedin Example 1, using RNA/probe mixes identical to those in Example 1.Following a 1 hour ligation with T4 ligase in the presence of ATP,NAD⁺-dependent mutant ligase and NAD⁺ may be added to the ligationreaction. For example, E. coli LigA point mutants Y22A, D32A and D36A,may be generated from wild-type LigA plasmid DNA by site-directedmutagenesis, using Stratagene's QuickChange kit or a similar product. Anamino-terminal truncated LigA construct may also be generated usingstandard PCR and subcloning approaches. One or more different mutants ofLigA, as described above, may be utilized in the present example as the“NAD⁺-dependent mutant ligase.” The ligation products will be resolvedand visualized as described in example 1.

Example 4 PCR of RNA-Templated Ligation of Padlock Probes

The experimental setup and timeline for the present Example 4 isillustrated in FIG. 3. This method uses padlock probes with the samesequences as listed above. Probe and RNA target concentrations in theannealing mix were 40 nM and 160 nM, respectively. Probe concentrationsrelative to RNA concentrations may be from 100:8, respectively to about10:8, respectively. Concentrations may be from pM to nM depending on theamount needed for signal detection or visualization. Charging of ligaseand ATP removal by apyrase treatment was performed as described above inExamples 1 and 2. Following 2 hours of ligation at 37° C., a sufficientamount of Exonuclease I (Exol) and Exonuclease III (ExoIII), i.e. about2 μl, were added and the reaction was further incubated at 37° C. for 20minutes, to digest unligated probe. Following inactivation of Exol andExoIII by incubation at 80° C. for 20 minutes, 5 μl of uracil-DNAglycosylase was added and the sample incubated at 37° C. for 15 minutes,to cleave the ligated circular padlock probe, followed by heatinactivation and 30 cycles of PCR.

1. A method for detecting a plurality of target nucleic acids from anucleic acids from a nucleic acid sample, comprising: a) contacting aDNA array-bound probe with: (i) a DNA interrogation probe, and (ii) anucleic acid sample comprising at least one RNA target nucleic acid,wherein the array-bound probe and the interrogation probe hybridize tothe target nucleic acid such that the 3′ end of the array-bound probeand the 5′ end of the interrogation probe are directly adjacent to oneanother and ligatable by DNA ligase, and wherein the interrogation probecontains a detectable label; b) adding an effective amount of ATP to asolution of DNA ligase, thereby charging the DNA ligase to form a DNAligase-AMP intermediate, c) depleting ATP from the solution of DNAligase-AMP intermediate; d) adding the solution of DNA ligase-AMPintermediate to step a), thereby ligating the 3′ end of the array-boundprobe to the 5′ end of the interrogation probe; and e) detecting theligated product.
 2. The method according to claim 1, wherein ATPdepletion is accomplished by adding an effective amount of apyrase inconditions which allow apyrase to catalytically convert ATP in solutionto ADP and/or AMP.
 3. The method according to claim 1, wherein ATPdepletion is accomplished by adding an effective amount of hexokinase inconditions which allow hexokinase to catalytically convert ATP insolution to ADP and/or AMP.
 4. The method according to claim 1, whereinthe DNA ligase is T4 DNA ligase.
 5. The method according to claim 2,wherein there is about 10 mU apyrase per μl of ATP depletion mix.
 6. Themethod according to claim 3, wherein there is about 0.4 U hexokinase perμl of ATP depletion mix.
 7. The method of claim 1 wherein theconcentration of ATP in the ligase charging mix is between 200 and 500mM.
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. A method of detectinga plurality of target nucleic acids from a nucleic acid sample,comprising: a) contacting an open circle DNA probe with an RNA nucleicacid sample comprising at least one target nucleic acid, wherein theopen circle probe comprises a 5′ end and a 3′ end, and wherein the opencircle probe contains a detectable label; b) adding an effective amountof ATP to a solution of DNA ligase, thereby charging the DNA ligase toform a DNA ligase-AMP intermediate, c) depleting ATP from the solutionof DNA ligase-AMP intermediate; d) adding the solution of DNA ligase-AMPintermediate to step a), thereby ligating the 5′ end of the open circleprobe to the 3′ end of the open circle probe; and e) detecting theligated product.
 12. The method according to claim 11, wherein ATPdepletion is accomplished by adding an effective amount of apyrase inconditions which allow apyrase to catalytically convert ATP in solutionto ADP and/or AMP.
 13. The method according to claim 11, wherein ATPdepletion is accomplished by adding an effective amount of hexokinase inconditions which allow hexokinase to catalytically convert ATP insolution to ADP and/or AMP.
 14. The method according to claim 11,wherein the DNA ligase is T4 DNA ligase.
 15. The method according toclaim 12, wherein the effective amount of apyrase is about 10 mU apyraseper μL.
 16. The method according to claim 13, wherein the effectiveamount of hexokinase is about 0.4 U hexokinase per μl.
 17. The method ofclaim 1 wherein the concentration of ATP in the ligase charging mix isbetween 200 and 500 mM.
 18. The method of claim 11 wherein theconcentration of ATP in the ligase charging mix is between 100 and 1000mM.
 19. The method of claim 11 wherein the concentration of ATP in theligase charging mix is between 0.1 and 200 mM.
 20. The method of claim11 wherein the concentration of ATP in the ligase charging mix isbetween 10 and 100 mM.